Protocols for collecting mouse PDL cells and bone marrow cells, differentiation, and data analysis

Summary Periodontal ligament cells (PDLCs) and macrophages in bone marrow cells have been widely used to investigate novel therapeutic agents to treat periodontitis. Here, we present a protocol for collecting primary mouse PDLCs and bone marrow cells. We detail steps for culturing and differentiation for both cell types and review data analysis for in vitro experiments using primary PDLCs and bone marrow cells. This protocol can be used to explore the impact of novel therapeutic agents using in vitro experiments. For complete details on the use and execution of this protocol, please refer to Sirisereephap et al.1

In addition to investigating therapeutic drugs for their impact on osteogenic differentiation, the examination the drugs effect on osteoclasts offers an avenue to explore the inhibitory effects on osteoclastogenesis, thereby impeding bone loss during the progression of periodontitis. 5 Osteoclastogenesis refers to the formation of osteoclasts, cells responsible for bone resorption.In in vitro studies focusing on osteoclastogenesis, the primary goal is usually to understand the factors and mechanisms influencing the differentiation and maturation of osteoclasts.Anticipated outcomes may differ based on distinct experimental conditions, cell types used, and the study objectives. 6proval to work with animals must be obtained from the appropriate authorities and the institution where the work will take place.Additionally, proper training in animal handling is mandatory.

Institutional permissions
Experiments received ethical approval from the Institutional Animal Care and Use Committee (IACUC) of Niigata University (approval no.SA00960).

Animal housing
Timing: 2 weeks 1.Obtain C57BL/6 wild-type mice aged between 6 to 8 weeks and allow a 2-week acclimation period to the experimental environment.Both genders are suitable for subsequent experiments; however, considering the potential impact of sex on the immune response, it is advisable to consistently employ sex-matched control groups or use mice of the same sex for all experiments.
CRITICAL: Ensure that experimental groups are matched in terms of both sex and age, as these variables can have an impact on the results.Additionally, when working with genetically modified mice, employ wild-type littermates as control groups to mitigate the influence of diverse genetic backgrounds as a potential explanation for observed differences between experimental groups.
Preparation for collecting primary mouse periodontal ligament cells (PDLCs) Timing: 2 h (for step 2) Timing: 1 h (for steps 3-6)   The day prior to the scheduled mouse sacrifice 2. Prepare and sterilize the surgical tools needed for sample collection (Figure 1).
On the day of sacrifice 3. Set the temperature of the water bath with agitation at 37 C, 200 rpm.4. Pre-chill the centrifuge to 4 C. 5. Prepare PDL complete medium.
CRITICAL: Filter medium through a 0.22 mm polyether sulfone (PES) membrane before use.
a. Equilibrate to room temperature 1 h before the experiment.6. Prepare the PDL digestion buffer.
CRITICAL: Prepare freshly before the experiment and filter the medium through a 0.22 mm polyether sulfone (PES) membrane before use.
a. Equilibrate to room temperature 1 h before the experiment.

Preparation to subculture and grow primary mouse PDLCs
Timing: 1 h On the subculture date 7. Pre-chill the centrifuge to 4 C. 8. Prepare PDL complete media.
CRITICAL: Filter the medium through a 0.22 mm polyether sulfone (PES) membrane before use.
a. Equilibrate to room temperature 1 h before the experiment.9. Thaw and equilibrate the ReagentPack Subculture Reagents (store at À20 C) to room temperature before the experiment for 1 h.
Optional: Thaw ReagentPack Subculture Reagents at 4 C 2 days before subculture day to minimize the time to equilibrate the reagents to room temperature.
Preparation for the osteogenic differentiation assay using primary mouse PDLCs Timing: 1 h 10.Prepare osteogenic differentiation medium (ODM).
CRITICAL: Filter the medium through a 0.22 mm polyether sulfone (PES) membrane before use.
a. Equilibrate to room temperature 1 h before the experiment.
Note: It is recommended to prepare ODM weekly to ensure the differentiation results.

Preparation for collecting primary mouse bone marrow cells
Timing: 2 h (for step 11) Timing: 20 min (for steps 12 and 13) The day prior to the sacrifice 11.Sterilize the instruments needed to collect samples.
On day of sacrifice 12. Pre-chill the centrifuge to 4 C. 13.Prepare the osteoclast supplemental medium.CRITICAL: Filter the medium through a 0.22 mm polyether sulfone (PES) membrane before use.
a. Equilibrate to room temperature 30 min before the experiment.
Preparation for the osteoclastogenesis assay using primary mouse bone marrow cells Timing: 15 min 14.Prepare the osteoclast differentiation medium.
CRITICAL: Filter the medium through a 0.22 mm polyether sulfone (PES) membrane before use.
a. Equilibrate to room temperature for 30 min before the experiment.

STEP-BY-STEP METHOD DETAILS
Primary mouse PDLCs collection Here, we describe steps to collect and culture primary mouse PDL cells before the experiment.
1. Euthanize the mice one by one and obtain the molars immediately after euthanasia.
Optional: After euthanization by isoflurane, perform transcardiac perfusion with PBS and 4% paraformaldehyde before separating the maxilla and mandible.

FBS 10% 1 mL
Total N/A 10 mL Always prepare fresh before each assay and store at 4 C before the experiment.Protocol CRITICAL: It is important to carefully separate a maxilla from the mouse head so as not to break the alveolar bone around the maxillary molars.
b. Carefully peel away and remove gingival tissue.c.Extract maxillary molars from the alveolar bone.
Note: It is recommended to perform this step under a Leica S6 stereomicroscope or under a high-magnification field.
CRITICAL: Do not forcefully extract tooth until the tooth is slightly mobile to avoid root fracture inside the alveolar bone.
d. Collect extracted teeth in 13 PBS (with Ca 2+ and Mg 2+ ) in a 12-well plate.e. Flush the teeth three times with a syringe containing 13 PBS and transfer them to a new well containing PBS.
CRITICAL: Do not let the teeth stay in PBS longer than 1 h as it will decrease the % of vital cells.
f. Check under the microscope to confirm that the PDL tissue is attached to the root surface (Figure 3).
Note: Upon visual inspection, the PDL tissues appear as a soft tissue exhibiting a light pink hue.This connective tissue intimately adheres to the tooth root surface and harbors a network of blood vessels.3. Incubate the teeth in 1.5 mL of PDL digestion buffer per 15 mL low-binding tube (STEMFULL Centrifuge Tube).
Note: STEMFULL is a centrifuge tube coated with an ultra-hydrophilic polymer that reduces cell loss by preventing nonspecific adherence to the tube walls.This coating ensures optimal cell recovery post-centrifugation.CRITICAL: Use collagen-coated plates to increase cell attachment.
b. Add 1 mL of cell suspension per well for a 12-well plate.c.Culture at 37 C in 5% CO 2 .d. Change the PDL complete medium every two days.

Note 1:
The average time to reach 80% confluent is one week.
Note 2: Discard the cell flask after 8 weeks of culturing as PDLCs are not differentiated.
7. Subculture when cells reach 80% confluent to increase cell number for the actual experiment.a. Follow the manufacturer's protocol.https://bioscience.lonza.com/lonza_bs/CH/en/download/product/asset/30937. b.Subculture from 12-well plate to T25 flask to increase cell number.

Osteogenic differentiation from primary mouse PDLCs
Timing: up to 28 days In this section, we describe steps to differentiate primary mouse PDL cells to osteoblast cells.
8. Obtain a confluent plate or flask of primary PDLCs.The representative images of primary mouse PDLC cultures are shown in Figure 4.
Note: Below is the protocol for a T25 (25 cm 2 ) flask culture.
a. Aspirate the culture medium from the culture vessel.b.Rinse the cells once with 5 mL pre-warmed HEPES-BSS.
CRITICAL: Skipping this step will lead to the failure of the trypsinization process to dissociate the attached cells, as the media contains proteins and calcium that neutralize the trypsin. 9Aspirate the HEPES-BSS from the flask.d.Add 2 mL of 0.25% Trypsin/EDTA solution.e. Allow the trypsin to cover the cell layer and incubate for 5 min at 37 C with CO 2 .f. Check the cell layer in the microscope.
Note: 90% of the cells should be detached at this time and appear circular in shape, floating in the solution.g.Gently tap the culture vessel against the palm of the hand to accelerate the cell dissociation from the vessel.
CRITICAL: If only a small number of cells detach, it is possible that the cells were not trypsinized for a sufficient duration.Wait for 30 s and tap again.Should detachment not occur, extend the wait not longer than 10 min and continue tapping every 30 s thereafter.
h. Neutralize the trypsin in the culture vessel with 4 mL of room temperature Trypsin Neutralizing Solution.i. Transfer the detached cells to a sterile 15 mL centrifuge tube.
Optional: A sterile 15 mL low-binding tube can be used to obtain a higher cell recovery.
i. Examine the empty flask under the microscope to confirm the harvest was successful.
ii.The remaining cells should be less than 5%.j.Centrifuge the harvested cells at 220 3 g for 5 min at 4 C.
i. Aspirate the supernatant.
ii. Add 1 mL of the PDL complete medium.
iii.Slowly resuspend the cell pellet with a micropipette.9. Seed the primary mouse PDLCs into a cell culture plate.
a. Calculate and adjust the live cell concentration to 2 3 10 4 cells per cm 2 in the desired volume for the experiment. 10te: The working volume for each well of a 48-well plate is 200 mL.
b. Add the adjusted cell suspension to the wells.c.Culture in the incubator for two days at 37 C with 5% CO 2 .
Note: Replace the PDL complete medium every two days and continue culture if the cells do not reach 80% confluency.
10. Culture the primary mouse PDLCS in the osteogenic differentiation medium.a. Aspirate the old medium from the culture plate.b.Replace with the osteogenic differentiation medium (ODM).c.Replace the medium every two days.d.Continue culture for 21 days. 1111.Culture the primary mouse PDLCS in the osteogenic mineralization medium.
a. Aspirate the old medium from the culture plate.b.Wash the cell culture wells with 13 PBS (with Ca 2+ and Mg 2+ ) once.c.Replace with the osteogenic mineralization medium (OMM).d.Replace the medium every two days.e. Continue culture for up to 28 days.
CRITICAL: Prolonged culture in the OMM will lead to abundant calcification, or calcification nodules are greater than 80% of culture area, in the cell culture wells.This causes difficulty to compare the calcification between experimental groups as the calcification area will be used to calculate the percentage of calcification area to represent the properties of each experimental agent to induce osteogenic differentiation.
12. Stain the cells for an analysis of osteogenic differentiation assay.a. Stain cells with Alizarin Red S (ARS) to visualize the calcification nodules from the differentiated osteoblasts (step-by-step explanation in the next protocol).
Note: Take histological images after the staining solution has been removed and replaced with DPBS solution.
ii. Obtain stereological images to calculate the percentage of the ALP-positive area (Figure 4).
CRITICAL: Air dry at room temperature overnight before capturing images.
Note: In the case of well plates larger than 48 wells, it is imperative to utilize a compact camera for capturing images of an entire well.This is essential because the minimum magnification of the Leica EZ4 Stereo Microscope is insufficient to encompass the entirety of a well, making it impractical for calculating the percentage of calcification based on ALP staining.
iii.Analyze the ALP-positive area using ImageJ software. 7v.Compare the ALP-positive area to the control group using Prism software.

Alizarin Red S (ARS) staining
Timing: 2 h Here, we describe steps to stain cell cultures with Alizarin Red S staining after undergoing osteogenic differentiation for 28 days.
Note: Below is the protocol for a 48-well plate.
Adjust the volume depending on the type of culture plate.Note: Take histological images after the DPBS solution has been added.
c. Examine the wells via a microscope and take photos using a stereo microscope (Figure 4).20.Obtain stereological images to calculate the percentage of calcification using ARS.
a. Aspirate remaining liquid.b.Dry overnight at room temperature.c.Take photos using a Leica EZ4 Stereo Microscope (Figure 4).
Note: In the case of well plates larger than 48 wells, it is imperative to utilize a compact camera for capturing images of an entire well.This is essential because the minimum magnification of the Leica EZ4 Stereo Microscope is insufficient to encompass the entirety of a well, making it impractical for calculating the percentage of calcification based on ARS staining.
d. Analyze the ARS-positive area using ImageJ software. 7.Compare the ARS-positive area to the control group using Prism software.

Primary mouse bone marrow cells collection Timing: 6 h
In this section, we describe steps to collect primary mouse bone marrow cells to obtain macrophages as the cell progenitors for osteoclasts.
Note: Below is the protocol for 96-well plate.
21. Euthanize mice by isoflurane or cervical dislocation.22. Collect femurs (Figure 5).a. Make a cut in the skin near the thigh.b.Separate the skin layers to find the femoral head.c.Remove the muscle around the femur.d.Rotate the femur around an axis to dissociate from a socket.e. Remove the remaining soft tissue.f.Cut both ends of the femur to make a cylinder appearance.g.In a petri dish, inject MEM-alpha medium, which is equilibrated to room temperature, to wash the femur by syringe.
Note: Use 2 mL of MEM-alpha medium per one femur.
h. Collect the fluid and transfer through a 40-mm cell strainer.i. Centrifuge at 400 3 g for 10 min at 4 C. j.Tilt the tube to discard the supernatant and aspirate any remaining supernatant.23.Add 2 mL of RBC lysis buffer.
a. Gently resuspend the cell pellet by pipetting.b.Incubate in RBC lysis buffer for 8 min at room temperature.
Note 1: For non-coated 96-well plate, the recommended number of live bone marrow cells for seeding is 10 5 cells per mL. 14te 2: The typical range of live cells from one femur is 2-4 3 10 6 cells.
25. Add the osteoclast supplemental medium to bone marrow cell suspension to adjust the cell concentration.26.Add 0.1 mL of the cell suspension per well (10 4 cell per well) of 96-well plate.27.Place the plate in the incubator for 3 h at 37 C in 5% CO 2 .

Timing: 6 days
Here, we describe the procedures for inducing the differentiation of primary mouse bone marrow cells into osteoclasts, along with the subsequent analytical methodology involving TRAP staining conducted at the conclusion of the assay.
28.After 3 h, aspirate the osteoclast supplemental medium.29.Wash with 13 PBS (with Ca 2+ and Mg 2+ ) once and replace with the osteoclast differentiation medium.30.Culture for 3 days at 37 C in 5% CO 2 .31.On the 3 rd day, check the cells under the microscope to confirm differentiation.
Note: Small multinucleated cells and/or cell fusion should be observed (Figure 6).32.Aspirate the old medium and replace it with the osteoclast differentiation medium.33.Continue culture for up to 7 days at 37 C in 5% CO 2 .34.On the 6 th day, check under the microscope to confirm differentiation.
Note: Take histological images after adding the DPBS solution.
c. Examine via a microscope and take photos using an EVOS M5000 microscope.37. Obtain stereological images to calculate the percentage of the TRAP-positive area (Figure 6).a. Aspirate the remaining liquid.b.Dry overnight at room temperature.c.Take photos using a Leica EZ4 Stereo Microscope.
Note: In the case of well plates larger than 48 wells, it is imperative to utilize a compact camera for capturing images of an entire well.This is essential because the minimum magnification of the Leica EZ4 Stereo Microscope is insufficient to encompass the entirety of a well, making it impractical for calculating the percentage of calcification based on ARS staining.
d. Analyze the TRAP-positive area using ImageJ software. 7.Compare the TRAP-positive area to the control group using Prism software.

EXPECTED OUTCOMES Primary mouse PDLCs
For six maxillary molars from one mouse, the total number of PDLCs should range 1-10 3 10 5 cells.
PDLCs can be combined from several mice and centrifuged prior to cell counting.The viability of each sample should be over 60%.Representative images of PDLCs in culture are shown in Figure 4.
Starting the molars collection from 3 mice, the live cells of collected PDLCs are suggested to seed on a single well of 12-well plate to expand the cells until it is confluent.PDLCs cultured in a 12-well plate should be 80% confluent within one week.Cells should be subcultured from a single 12-well plate to T25 flask to expand the cell number.A T25 flask should be confluent within one week after subculture from one well of a 12-well plate.One confluent flask of T25 flask should range 1-2 3 10 6 cells and can be used for two plates of the 48-well plate.For 48-well plate, the appropriate number of cells for seeding is 5 3 10 3 cells/well.
Characterization of mineralization and differentiated osteoblasts from primary mouse PDLCs after osteogenic differentiation Anticipated outcomes from in vitro experiments focusing on osteoblast differentiation from primary mouse PDLCs involve several key observations.The outcomes should include the formation of osteoblasts, characterized by their ability to produce mineralized extracellular matrix and their enzymatic activity of alkaline phosphatase.In this experiment, the formation of mineralized nodules detected by ARS staining is key to successful osteoblast differentiation.The anticipated outcome of ARS staining in the context of osteoblast differentiation involves the visualization and quantification of mineralized matrix formation, demonstrating complete osteogenic differentiation.Alizarin red is a dye that binds to calcium ions, enabling the detection of mineralized nodules formed by osteoblasts.The staining results in the formation of a distinctive red color in areas where calcium deposits have accumulated, reflecting the presence of a mineralized extracellular matrix produced by differentiated osteoblasts. 15This outcome is critical for assessing the effectiveness of osteoblast differentiation protocols and for studying factors influencing bone mineralization.The representative images of the calcification nodules after the osteogenic differentiation are shown in Figure 4.
ALP staining is a widely utilized method for evaluating osteogenic differentiation.The anticipated result of alkaline phosphatase staining in osteoblasts entails visualizing enzymatic activity, particularly alkaline phosphatase activity that is important enzyme for bone mineralization.This staining produces a colored substrate at sites exhibiting alkaline phosphatase activity within the osteoblasts.Alkaline phosphatase's role in the mineralization process involves hydrolyzing inorganic pyrophosphate, a mineralization inhibitor, emphasizing its importance in bone development.Detection and activity of alkaline phosphatase in osteoblasts indicates the generation of a mineralized extracellular matrix. 16The expected outcome of ALP staining is the identification of stained areas in osteoblasts, confirming their differentiation and active participation in bone mineralization.The representative images of ALP-positive cells after osteogenic differentiation are shown in Figure 4.
Characterization of differentiated osteoclasts from primary mouse bone marrow cells When using primary mouse bone marrow cells, common findings, and observations in osteoclastogenesis experiments should include the formation of giant multinucleated cells or the generation of osteoclasts with multiple nuclei.The formation of giant multinucleated cells is a key outcome in successful osteoclastogenesis, as these cells are generally characterized by their substantial, multinucleated nature.Moreover, successful experiments should show evidence of cell fusion during the differentiation process, as osteoclastogenesis involves the fusion of precursor cells to form multinucleated osteoclasts.The representative images of osteoclast differentiation are shown in Figure 6.
TRAP staining is used to characterize osteoclasts via the activity of tartrate-resistant acid phosphatase.When treated with staining reagents, osteoclasts manifest a cytoplasmic stain in hues of red or purple, indicating the enzymatic activity associated with TRAP.This particular method of staining shows the activity of the TRAP enzyme, which is active under acidic conditions, facilitating the localization of osteoclasts in close proximity to sites of bone resorption. 17The resulting staining outcome not only aids in qualitatively assessing the distribution of osteoclasts but also provides valuable insights into TRAP-related enzymatic activity linked with bone remodeling.Furthermore, TRAP staining offers the quantitative data that holds significance in studies focused on bone metabolism.The representative images of TRAP-positive cells are shown in Figure 6.

LIMITATIONS
When collecting PDLCs from multiple donors, variability may exist between batches of harvested cells, resulting in varying degrees of growth efficiency.After starting cell cultures using diverse sets of collected PDLCs, only those wells showing optimal growth characteristics are retained for prolonged cultivation and subsequently subjected to the osteogenic differentiation assay.If starting with six maxillary molars derived from a single mouse, it is expected that only enough PDLCs will be collected for one well of a 12-well plate to reach 80% confluency within one week.
Due to the extended duration of PDLC culture, spanning a requisite period of two months to achieve the necessary cell density and osteoblast differentiation, it is imperative to exercise meticulous care in handling the cell cultures.It is essential to maintain sterile conditions and vigilantly monitor the cultures for any indications of contamination.In the event that contamination is observed in any well, the optimal course of action is to discard the entire plate.This precautionary measure aims to curb the proliferation of contaminants and prevent the preservation of contaminated cells, which could potentially give rise to secondary contamination in subsequent processes.
In addition, cellular heterogeneity poses a significant challenge in the culture of primary mouse macrophages in bone marrow cells, underscoring the importance of establishing rigorous control over in vitro conditions to thoroughly delineate macrophage responses to differentiate into osteoclasts. 18 summary, the existing protocols provide guidelines for sample collection, cell culture techniques, and staining procedures to visualize data for one of the parameters analyzed in the successful differentiation assay.Nevertheless, it is strongly advised to conduct molecular biology assays to validate the gene and protein expression of specific cell markers following cell differentiation.

TROUBLESHOOTING Problem 1
It is difficult to extract mouse maxillary molars, especially the first maxillary molar.(step 2 of the primary PDLCs collection).

Potential solution
Introduce a 20-gauge needle between the first and second molars to facilitate tooth loosening. 9For the first molar, use the tip of Dumont forceps to establish a gap between the tooth and alveolar bone.Then, firmly hold the tooth and gently luxate it.Upon achieving mobility, securely grasp the tooth and extract it.Caution should be exercised, as mishandling may result in tooth flip, necessitating the removal of any contaminated tooth from the collected samples.

Problem 2
The number of collected PDLCs is low and not sufficient for cell culture (step 5 of the primary PDLCs collection).

Potential solution
Generally, one mouse, from which six maxillary molars can be collected, is sufficient for the PDLCs to be seeded in a single well of a 12-well plate.Use periodontal microsurgery instruments or surgical blade for scraping of PDL tissues from the root surfaces. 8,19Moreover, careful attention to timing is essential during the collection of PDLCs, an operation exceeding 1 h has been observed to lead to a cell viability below 50%.

Problem 3
The PDLCs detach from the culture vessel during culture and/or experiment (step 7 and 10 of the primary PDLC culture and differentiation).

Potential solution
Use a collagen-coated plate or flask to ensure proper attachment of PDLCs to the surface during cell culture.Exercise caution when aspirating media from the cell culture to avoid touching the bottom of the well, as this could disrupt the cell layer and result in PDLC detachment.When adding medium to a well, position the pipette tip at the side wall and slowly depress the pipette plunger to gently introduce the medium into the well.

Problem 4
The mineralized nodules are not observed in cell culture after ARS staining.(step 18 of ARS staining after osteogenic differentiation).STAR Protocols 5, 103162, September 20, 2024

Protocol Potential solution
Use the PDLCs for osteogenic differentiation or supplementary experiments within two months postcollection.Extended culture of PDLCs may result in the attenuation of their osteoblastic differentiation potential.Additionally, it is imperative to prepare fresh ODM and OMM weekly to maintain the nutrients required for cell growth during the assay.The appearance of calcification nodules, typically manifested as white spots, is commonly observed around the second week of osteogenic differentiation.

Problem 5
The multinucleated cells are not observed on the 3 rd day of the assay.(step 31 of the primary mouse bone marrow cells collection).

Potential solution
Check the quality of bone marrow cells collected during collection process.Exercise caution not to over-incubate the cell pellet in the ACK lysis buffer, as this may lead to a decrease in cell viability.Nevertheless, insufficient incubation can result in inadequate red blood cell lysis, diminishing osteoclastogenesis due to interreference from RBCs present in the cell culture. 14It is essential to prepare fresh osteoclast-supplemental and osteoclast differentiation media for each experiment, prior to media changes, and to equilibrate the media to 37 C before adding it to the cells.Additionally, appropriate reconstitution and proper storage of crucial reagents, such as M-CSF or recombinant RANK-L, are imperative to maintain protein function and ensure osteoclastogenesis results.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Tomoki Maekawa (Maekawa-t@dent.niigata-u.ac.jp).

Technical contact
Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Kridtapat Sirisereephap (Kridtapat.s@chula.ac.th).

Materials availability
Cell culture plates generated in this study belong to the collections of The Center for Advanced Oral Science, Graduate School of Medical and Dental Sciences, Niigata University and can be studied upon request.

Figure 1 .
Figure 1.Tools for collecting PDLCs and primary bone marrow cells from mouse (A) From left to right: 26G needle, 1 mL syringe, no. 10 scalpel blade, curved forceps (Perry), suture-tying forceps, Dumont forceps, and surgical scissors.(B) The actual setup for sample collection.The styrene foam is covered with aluminum foil, and a G-26 needle is used to position the mouse.Autoclave the surgical instruments before the experiment at 121 C for 20 min.

Figure 2 .
Figure 2. Surgical procedure to extract mouse maxillary molars (A) Position the mouse in a supine posture with its head oriented towards the operator.(B) Incise both sides of the cheek to facilitate access to the maxillary molars.(C) The optimal access for initiating the molar extraction process.(D) Remove the palatal gingiva and soft tissue surrounding the molars.(E) Tooth extraction positioning: On the left side, use an instrument to stabilize the maxilla; on the right side, use Dumont forceps to luxate the tooth and extract it from the tooth socket.(F) Extracted sockets following the removal of maxillary left molars.

Figure 3 .
Figure 3.The molar after incubation in the PDL digestion buffer (A) The extracted maxillary second molar.(B) Incubate the extracted molars in PBS.(C and D) The extracted maxillary second molar under the Leica EZ4 stereo microscope.(C) The extracted molar with attached PDL tissues on the root surface (red arrow).(D) The extracted molar after incubation in the PDL digestion buffer.No PDL tissue is observed after enzyme digestion.Scale bar: 0.5 mm.

8 a.
Place the tubes in the incubator with agitation at 200 rpm for 20 min at 37 C. b.Add 15 mL of 0.5 M EDTA to each tube.c.Place the tubes in the incubator with agitation at 200 rpm for 10 min at 37 C. d.Add 9 mL of washing buffer that is equilibrated to room temperature (PBS containing 2% FBS).e. Centrifuge at 400 3 g for 8 min at 4 C. 4. Resuspend the cell pellet in 2 mL of washing buffer.a. Filter the supernatant through a 70-mm cell strainer (BMS, BC-AMS-17001).b.Centrifuge at 320 3 g for 5 min at 4 C. 5. Resuspend the cell pellet in 1 mL of washing buffer.a. Count the number of living cells.Note: The average number of live cells from a single second molar is 4 3 10 3 cells.b.Add PDL complete medium to adjust the cell concentration at 10 4 cells per mL.6. Seed the cell in a culture vessel.a. Seed the cells at 10 4 cells per well in a 12-well plate.

Figure 4 .
Figure 4. Osteogenic differentiation of primary mouse PDLCs (A) Primary PDLCs at Day 2 after collection.The attached PDLCs predominantly maintain a circular shape.(B) Primary PDLCs at Day 6 after collection.The majority of cells exhibit a spindle-shaped morphology (fibroblast-like cells, red dots).(C-F) Differentiated cells from PDLCs at Day 28 of the osteogenic differentiation assay.(C, D) Cell cultures were stained with ALP stain.(C) Microscopic image showing ALP-positive cells from the cell cultures.(D) The entire well of cultured cells after ALP staining.Used for calculating the percentage of ALP-positive area.(E, F) Cell cultures were stained with ARS.(E) Microscopic image showing mineralized nodules visualized by ARS staining.(F) The entire well of the cultured cells after ARS staining.Used for calculating the percentage of the mineralization area.Scale bar: 300 mm.

Figure 6 .
Figure 6.Procedure to collect primary mouse bone marrow cells (A) Primary bone marrow cells after 3 h incubation in osteoclast supplemental medium.The cells predominantly maintain a small circular shape.(B) Osteoclasts differentiated from primary bone marrow cells on Day 3 after collection.Multinucleated cells and cell fusion should be observed at this time point (red dots).(C) Osteoclasts differentiated from primary bone marrow cells on Day 6.Giant multinucleated cells were observed (red dots) before staining with a TRAP staining kit.(D and E) Cell cultures after staining with a TRAP staining kit.(D) Microscopic image showing giant multinucleated cells in cell culture.These multinucleated cells manifest a cytoplasmic stain in hues of red or purple when treated with reagents from the TRAP staining kit, indicative of the TRAP enzymatic activity.(E) The entire well of mouse bone marrow cells after TRAP staining.Used for calculating the percentage of the TRAP-positive area.Scale bar: 300 mm.
and T. Maekawa; investigation, K.S. and M.D.C.S.; resources, T. Maekawa and T. Maeda; writingoriginal draft preparation, K.S.; writing -review and editing, K.S., M.D.C.S., A.L.R., and T. Maekawa; project administration, T. Maekawa; funding acquisition, T. Maekawa.All authors have read and agreed to the published version of the manuscript.

TABLE REAGENT
Use the prepared ODM and add Dexamethasone (final as 10 nM).